Collagen-coated Tissue-based Membranes

ABSTRACT

A collagen-coated tissue based membrane that is smooth on both sides so as to inhibit cell and tissue adhesion. Also disclosed are methods for making a collagen-coated tissue-based membrane.

BACKGROUND

Biocompatible membranes can be prepared by several different methods. Inone method, purified collagen fibers extracted from collagen-richtissues (e.g., tendon and dermis) are used to reconstitute a membrane,as described in U.S. Pat. Nos. 6,391,333; 6,599,524; and 7,807,192. Analternative method relies on the use of a tissue-derived membrane (e.g.,pericardium, peritoneum, and small intestine submucosa) as a startingmaterial, as described in U.S. Pat. Nos. 5,837,278 and 5,993,844.

Tissue-derived membranes such as pericardium and peritoneum typicallycontain two sides. The side that is in contact with internal organs andbody fluid is known as the visceral, or serous, layer, while the sidethat is in contact with the abdominal or heart walls is termed theparietal layer. The visceral layer, which is smooth, does not adhere toadjacent body tissue. By contrast, the parietal layer is integrated withthe tissue adjacent to it. The parietal layer must be mechanicallyseparated from the adjacent body wall tissue in order to remove atissue-based membrane from the body. The parietal layer of atissue-based membrane torn from the integrated soft tissue of the bodywall is covered with fibrous material. This fiber-covered surface of theparietal layer is adhesive, favoring cell adhesion and tissue growth.The fibrous material on the parietal side of membranes isolated fromanimals can be easily identified under microscopy, such as scanningelectron microscopy.

Biocompatible membranes are often used in medical and dental surgeries.At times, it is necessary to use a membrane that is not adhesive oneither side so as to avoid any adhesion to the surrounding tissue. Forexample, a non-adhesive membrane can be used for surgical repair of torndural membranes, as well as for dental surgery requiring guided tissuerepair.

The need exists for a tissue-based biocompatible membrane that can beused in situations where cell adhesion to the membrane is undesirable.

SUMMARY

The main objective of the present invention is to provide the surgicalcommunity with a biocompatible tissue-based membrane that is minimallyadhesive on both sides for use in tissue repair and regenerationapplications.

Thus, one aspect of this invention relates to a tissue-based coatedmembrane that includes a serous membrane having a fibrous surface coatedwith a layer of collagen and an uncoated smooth surface such that boththe coated fibrous surface and the uncoated smooth surface adhere poorlyto cells and tissues. The serous membrane can be, e.g., pericardium,peritoneum, amnion, small intestine submucosa, pleural, or vaginaltunics. In a preferred embodiment, the serous membrane is pericardium orperitoneum. In another preferred embodiment, the fibrous surface of theserous membrane is coated with fiber-forming collagen. The fiber-formingcollagen can be type I collagen, type II collagen, type III collagen, ora combination of two or more of these three types.

Another aspect of this invention relates to a method for preparing abiocompatible collagen-coated serous membrane. The method includes astep in which the fibrous side of the serous membrane is scraped toremove adhering fibers. The scraped serous membrane is then treated toremove cells, lipids, and extractable blood and non-collagenousmolecules, followed by freeze-drying of the treated serous membrane. Thefibrous side of the treated serous membrane is compressed and thencoated with a collagen. Finally, the collagen-coated serous membrane isexposed to a crosslinking agent to effect crosslinking. In anembodiment, the serous membrane is rinsed with a dehydrating agent priorto the scraping step to allow for more efficient removal of fibersadhering to the fibrous side of the membrane. The dehydrating agent canbe a small organic molecule such as acetone or an alcohol. In anotherembodiment, the fibrous side of the treated serous membrane is coatedwith a fiber-forming collagen that can be type I collagen, type IIcollagen, type III collagen, or a combination thereof. It is preferredthat the collagen is type I collagen. The collagen can be coated ontothe fibrous side of the treated serous membrane by spraying, brushing,or dipping. Preferably, the collagen is sprayed onto the fibrous side ofthe serous membrane.

Also provided is a biocompatible collagen-coated serous membraneprepared by the above-described method.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and from the claims.

DETAILED DESCRIPTION

This invention relates to a method for preparing a tissue-based coatedmembrane resulting in a membrane that is smooth on both sides so as tominimize tissue adhesion.

The method for producing a smooth tissue-based coated membrane includesthe following six key steps. First, the fibrous, i.e., parietal, side ofan isolated tissue-based membrane is scraped to remove adhering fibers.Second, the tissue-based membrane is processed with an enzyme andchemicals to remove cells, lipids, and extractable blood andnon-collagenous molecules. Third, the processed tissue-based membrane isfreeze dried. Fourth, the fibrous side of the tissue-based membrane iscompressed. Fifth, the compressed fibrous side of the tissue-basedmembrane is coated with collagen. Finally the collagen coatedtissue-based membrane is exposed to a crosslinking agent. Tissue-basedmembranes, e.g., bovine or porcine peritoneum or pericardium, providedby commercial suppliers generally contain fibers that adhere to theparietal side of the membrane, resulting from mechanical separation ofthe tissue-based membrane from neighboring tissues.

Depending on the location of the membrane in the body and the harvestingtechnique, the amount of adhering fibers varies. Removal of the adheringfibers is important to minimize potential surface irregularities.Applicants disclose that rinsing the membrane with a dehydrating agent,such as a low molecular weight organic compound or an alcohol, canfacilitate the removal of adhering fibers, as partial dehydrationrenders the fibers stiff and more easily removed by scraping or othermechanical means. For example, the membrane can be rinsed with acetone,methanol, ethanol, or isopropanol.

The next step in processing the tissue-based membrane is to remove asmuch of the loosely associated proteins, cell debris, DNA materials,blood associated proteins, water soluble moieties, and lipids aspossible using a combination of enzymes and chemicals. A series ofsequential chemical treatments and treatment with an enzyme areperformed to remove non-collagenous moieties from the tissue, to removeDNA molecules from cells and cell nuclei from the tissue, and toinactivate undesired potentially harmful viruses from the tissue. Thechemicals and enzyme used for carrying out this step include 0.1-3%octylphenol ethoxylate (TRITON X100 ™, 2-8 hour treatment), DNAse (2-18hour treatment), 1 M NaOH (5-10 hour treatment), 0.5 M HCl (5-10 hourtreatment), 1 M NaCl (24-48 hour treatment), isopropyl alcohol (24-72hour treatment), and water (5-24 hour treatment).

The enzyme and chemical treatments described above remove most of thenon-collagenous proteins, lipids, and cell-associated moieties from thetissue. Yet, the tissue retains its native structure and associatedhandling and mechanical properties.

The clean, treated tissue is then freeze-dried under conditions wellknown in the art, such as those disclosed in U.S. Pat, No, 6,391,333,the content of which is hereby incorporated by reference in itsentirety. Typically, for thin membranes, freeze drying is accomplishedby incubation of the membrane at −20° C. for 24-48 hours at a vacuumbelow 300 Ton, followed by drying at 20° C. for 8-24 hours.

After the above treatments, the freeze-dried membrane is still rough onits parietal, i.e., fibrous, side. In order to make the fibrous sidesmooth, it is coated with collagen. The membrane is prepared for coatingin two steps. First, it is softened by incubation in a humidifiedchamber. Exposure of the membrane to a relative humidity greater than90% for 1 to 4 hours is normally sufficient to soften the membrane forfurther processing. Second, the softened membrane is then compressed bypassing a roller over the fibrous side of the membrane. The roller hassufficient weight to completely flatten and smooth any surface fibersstill adhering to the fibrous side of the membrane. For example, theweight of the roller can be between 2 and 8 kg.

The coating of the membrane can be accomplished by spraying the surfacewith acid dispersed collagen fibers (e.g., dispersed in lactic acid) ata pH of 2.3-2.5, or alkaline dispersed collagen fibers (e.g., dispersedin NaOH) at a pH of 11 to 12. Any commercial sprayer (e.g., BadgerAirbrush Model 150) can be used to spray the dispersed collagen fibersonto the fibrous side of the membrane. Spraying can be performed at10-60 psi using a nozzle size of 0.1-1.0 mm. The concentration ofcollagen fibers in the spraying solution ranges from about 0.05% toabout 0.1% (w/v) such that the collagen is sprayed in the form of a mistthat coats the membrane surface uniformly despite any microscopicirregularities that may exist. A single layer or multiple layers of acollagen coating can be applied via this spraying technique. Typically,the thickness of the collagen coating is in the range of 5-50 μm.

Alternatively, the fibrous surface can be coated with an acid dispersedcollagen using a brush. The concentration of collagen used for brushcoating can be significantly higher than that used for spraying, rangingfrom about 0.1% to about 0.9% (w/v). The collagen coating applied usinga brush is significantly thicker than that applied by spraying, rangingfrom about 50 μm to about 100 μm.

Further, the membrane can be dipped into a collagen dispersion in orderto coat the fibrous surface of the membrane.

Irrespective of the collagen coating technique employed, the coatedmembrane is dried so as to more strongly integrate collagen with themembrane surface.

The coated membranes can be crosslinked using a crosslinking agent thatis well known in the art. Crosslinking can be conducted in a solutioncontaining a crosslinking agent (e.g., glutaraldehyde, formaldehyde) orby exposing the coated membrane to the vapor of a crosslinking agent(e.g., formaldehyde vapor). The extent of crosslinking in solution is afunction of the concentration of the crosslinking agent, the temperatureof the solution, and the time of crosslinking. For example, crosslinkingcan be accomplished by exposing the coated membrane to a 0.5%formaldehyde solution for 5 hours at room temperature. The crosslinkingcondition can significantly affect the characteristics of the membrane.For example, a high concentration of the crosslinking agent and a longercrosslinking time will result in a less conformable membrane having aslow rate of in vivo resorption. Conversely, in order to prepare a moreconformable membrane, a low concentration of a crosslinking agent ispreferred. The same principle applies to the vapor crosslinking, where alower vapor pressure of a crosslinking agent will produce a moreconformable membrane. The crosslinked membrane can be rinsed or exposedto air in order to reduce the amount of any crosslinking agent residueto a level that is acceptable for human implantation.

Without further elaboration, it is believed that the above descriptionhas adequately enabled the present invention. The following threeexamples are, therefore, to be construed as merely illustrative, and notlimitative of the remainder of the disclosure in any way whatsoever.

EXAMPLE 1 Collagen-Coated Bovine Pericardium Membrane Preparation ofPurified Tendon Collagen

The fat and fascia of a bovine flexor tendon were carefully removed andthe tendon washed with water. The cleaned tendon was frozen andcomminuted by slicing into 0.5 mm slices with a slicer. The slicedtendon was first extracted in purified water at room temperature for 24hours. After decanting the water, a solution of 0.2 M HCl in 0.5 MNa₂SO₄ was added and the tendon slices were extracted at roomtemperature for 24 hours. The acid solution was neutralized by adding 3M NaOH to raise the pH to approximately 7. The tendon slices wereextracted in this first neutralized salt solution for 18 hours, afterwhich the solution was removed by decanting. The acid-extracted tendonwas then further extracted with 0.75 M NaOH in 1 M Na₂SO₄ at roomtemperature for 24 hours. This base solution was neutralized to a pH ofapproximately 7 by adding 3M HCl. The tendon slices were extracted inthis second neutralized salt solution for 18 hours, after which thesolution was removed by decanting. After the two just-mentionedneutralized salt extractions, the tendon slices were rinsed withpurified water 4 times in order to remove any residual salts. Theextracted, rinsed tendon was then defatted with 99% isopropanol for 8hours at room temperature. The isopropanol was decanted and a secondequal volume of 99% isopropanol was added to further extract the tendonslices for 18 hours at room temperature.

All extraction steps described above were performed under constantagitation. Further, all extractions, with the exception of theisopropanol extractions, were performed using 5 ml of extractingsolution per gram of tendon processed. The isopropanol extractions wereperformed using 3 ml of isopropanol per gram of tendon processed.

The defatted tendon was then dried under a clean air hood. The processedtendon material, consisting primarily of purified fibrillar collagen,was stored dry at room temperature for later use.

Preparation of Acid Dispersed Collagen Fibers

A 0.1% (w/v) acidic collagen dispersion was prepared by swelling 1 g ofpurified fibrillar collagen in one liter of 0.07 M Lactic acid at 4° C.in a refrigerator overnight. The swollen fibers were then homogenizedfor 60 seconds using a Silverson homogenizer and filtered through a 50mesh stainless steel filter. The acidic dispersion was then stored inthe refrigerator for later use.

Purification of Bovine Pericardium Membrane

Fat and extraneous tissue were mechanically removed from the raw bovinepericardium tissue and washed with water. The pre-cleaned bovinepericardium was first washed in purified water at room temperature for 2hours. The water was decanted, 0.1% TRITON X-100™/DNase was added, andthe pericardium was extracted for two hours at room temperature. Theamount of DNase used was 4 units of activity per cm² of membrane. Thesolution was decanted and the bovine pericardium was then rinsed withpurified water for 2 hours. The detergent/enzyme-extracted bovinepericardium was then defatted by incubation with 99% isopropanol threetimes for 6, 18, and 24 hours at room temperature. The defatted bovinepericardium was then extracted in 0.5 M HCl in 0.5 M Na₂SO₄ for 6 hoursat room temperature. This acidic solution was neutralized by adding 3 MNaOH to a pH of approximately 7. The bovine pericardium was thenextracted in the neutralized salt solution for 18 hours and the solutionwas decanted. The acid extracted bovine pericardium was then furtherextracted in 1 M NaOH in 1.2 M Na₂SO₄ at room temperature for 6 hours.This basic solution was neutralized by adding 3 M HCl to a pH ofapproximately 7. The bovine pericardium was then extracted in theneutralized salt solution for 18 hours and the solution was decanted.After the salt extractions, the bovine pericardium was washed 4 timeswith purified water to remove the residual salts associated with thepurified bovine pericardium. All extraction steps were performed underconstant agitation.

All extractions, with the exception of the isopropanol extractions, wereperformed using 3 ml of extracting solution per cm² of bovinepericardium processed. The isopropanol extractions were performed using2.7 ml of isopropanol per cm² of bovine pericardium processed.

The purified bovine pericardium was then freeze-dried and stored untillater use.

Coating of the Purified Bovine Pericardium With Acid Dispersed Collagen

The freeze-dried purified bovine pericardium was humidified in ahumidification tank for approximately 30-60 minutes at a relativehumidity of 90-100%. The fibrous (parietal) side of the membrane wasthen compressed using a roller under light pressure in a single uniformdirection to flatten the fibers along that direction. The roller weighed4 kg, and the light pressure was provided solely by the weight of theroller. The membrane was placed in a clean air hood to dry for a minimumof 15 minutes. The membrane was then placed in a holding frame and,using an air-brush gun (Badger Airbrush Model 150), the membrane wassprayed with a 0.1% (w/v) acidic collagen dispersion for 30 seconds. Thecollagen dispersion was sprayed at 40 psi through a 0.3 mm nozzle. Thesprayed membrane was then placed into a glass dish and lightlycrosslinked with a 0.001% glutaraldehyde solution for 3 hours. Thecrosslinked membrane was then rinsed with purified water andfreeze-dried again.

Characterization of the collagen-coated bovine pericardium membraneScanning electron microscopy (SEM) was performed on collagen-coated andnon-coated samples at 50× magnification using a scanning electronmicroscope (JEOL Ltd. Model JSM 6100 SEM). The samples were mounted onan aluminum planchet using adhesive carbon tape. Approximately 500angstroms of pure gold was sputtered onto the samples to improveelectron beam conduction and prevent sample charging.

The fibrous (parietal) side of a bovine pericardial membrane has a roughappearance as visualized under SEM as a result of randomly-orientedfibers on the surface of the membrane. A smooth surface is seen underSEM after coating the parietal surface of the bovine pericardialmembrane with collagen following the procedure described above. Thesmooth (visceral) non-coated side of the bovine pericardial membrane issimilar in appearance to the coated fibrous side.

EXAMPLE 2 Collagen-Coated Porcine Peritoneum Membrane

Fat and extraneous tissue were mechanically removed from the porcineperitoneum and washed with water. The pre-cleaned porcine peritoneum wasfirst washed in purified water at room temperature for 2 hours. Thewater was decanted and 3% TRITON X100™ was added to the peritoneum andincubated for 7 hours at room temperature. The TRITON X100™ solution wasdecanted and the peritoneum then incubated in a DNase solution for 18hours. The amount of DNase used was 8 units of activity per cm² ofmembrane. The detergent and enzyme extracted porcine peritoneum wasdefatted by incubating it with 99% isopropanol three times for 3, 3, 18,and 24 hours at room temperature. The defatted porcine peritoneum wasextracted in 0.5 M HCl in 0.5 M Na₂SO₄ for 6 hours at room temperature.This acidic solution was neutralized using 3 M NaOH to a pH ofapproximately 7. The porcine peritoneum was further extracted in theneutralized salt solution for 18 hours and the solution was decanted.The acid extracted porcine peritoneum was then extracted in 1 M NaOH in1.2 M Na₂SO₄ at room temperature for 6 hours. This basic solution wasneutralized using 3 M HCl to a pH of approximately 7. The porcineperitoneum was further extracted in the neutralized salt solution for 18hours and the solution was decanted. After the salt extractions, theporcine peritoneum was washed 4 times with purified water to remove theresidual salts associated with the purified porcine peritoneum.

All extractions, with the exception of the isopropanol extractions, wereperformed using 3 ml of extracting solution per cm² of porcineperitoneum processed. The isopropanol extractions were performed using2.7 ml of isopropanol per cm² of porcine peritoneum processed.

The purified porcine peritoneum was freeze-dried and stored until lateruse.

The purified porcine peritoneum membrane was coated with acid dispersedcollagen as described above for the bovine pericardium membrane.

EXAMPLE 3 Coating of Bovine Pericardium With Alkaline Dispersed CollagenFibers

A 0.1% (w/v) alkaline collagen dispersion was prepared by swelling 1 gof fibrillar collagen purified as described above in one liter of 0.01 MNaOH at 4° C. in a refrigerator overnight. The swollen fibers were thenhomogenized for 90 seconds using a Silverson homogenizer, after whichthey were filtered through a 50 mesh stainless steel filter. Thealkaline dispersion was then stored in the refrigerator for later use.

A bovine pericardium membrane is purified as described above. Alkalinedispersed collagen fibers are coated onto the purified membrane asdescribed above for acid dispersed collagen fibers.

EXAMPLE 4 Coating of Porcine Peritoneum With Alkaline Dispersed CollagenFibers

A 0.1% (w/v) alkaline collagen dispersion was prepared by swelling 1 gof fibrillar collagen purified as described above in one liter of 0.01 MNaOH at 4° C. in a refrigerator overnight. The swollen fibers were thenhomogenized for 90 seconds using a Silverson homogenizer, after whichthey were filtered through a 50 mesh stainless steel filter. Thealkaline dispersion was then stored in the refrigerator for later use.

A porcine peritoneum membrane is purified as described above. Alkalinedispersed collagen fibers are coated onto the purified membrane asdescribed above for acid dispersed collagen fibers.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features. From the above description, one skilled in the art caneasily ascertain the essential characteristics of the present invention,and without departing from the spirit and scope thereof, can makevarious changes and modifications of the invention to adapt it tovarious usages and conditions. Thus, other embodiments are also withinthe scope of the following claims.

What is claimed is:
 1. A tissue-based coated membrane, comprising a serous membrane and a collagen coating, wherein the serous membrane has a fibrous surface and a smooth surface, the collagen coating is present on the fibrous surface, and both the collagen-coated fibrous surface and the smooth surface adhere poorly to cells and tissues.
 2. The tissue-based membrane of claim 1, wherein the serous membrane is pericardium, peritoneum, amnion, small intestine submucosa, pleural, or vaginal tunics.
 3. The tissue-based membrane of claim 1, wherein the collagen is fiber-forming collagen.
 4. The tissue-based membrane of claim 3, wherein the fiber-forming collagen is type I collagen, type II collagen, type III collagen, or a combination thereof.
 5. The tissue-based membrane of claim 4, wherein the fiber-forming collagen is type I collagen.
 6. The tissue-based membrane of claim 5, wherein the serous membrane is pericardium or peritoneum.
 7. A method for preparing a biocompatible collagen-coated serous membrane, the method comprising: scraping a fibrous side of a serous membrane to remove adhering fibers; treating the serous membrane to remove cells, lipids, and extractable blood and non-collagenous molecules; freeze-drying the treated serous membrane; compressing the fibrous side of the serous membrane; coating the fibrous side of the serous membrane with a collagen; and exposing the collagen coated serous membrane to a crosslinking agent to effect crosslinking.
 8. The method of claim 7, wherein the serous membrane is rinsed with a dehydrating agent prior to the scraping step.
 9. The method of claim 8, wherein the dehydrating agent is an alcohol.
 10. The method of claim 7, wherein the serous membrane is pericardium, peritoneum, amnion, small intestine submucosa, pleural, or vaginal tunics.
 11. The method of claim 7, wherein the collagen is fiber-forming collagen.
 12. The method of claim 11, wherein the fiber-forming collagen is type I collagen, type II collagen, type III collagen, or a combination thereof.
 13. The method of claim 12, wherein the fiber-forming collagen is type I collagen.
 14. The method of claim 7, wherein the coating is accomplished by spraying the collagen onto the fibrous side of the serous membrane.
 15. The method of claim 7, wherein the coating is accomplished by brushing the collagen onto the fibrous side of the serous membrane.
 16. The method of claim 7, wherein the coating is accomplished by dipping the fibrous side of the serous membrane into the collagen.
 17. The method of claim 14, wherein the serous membrane is pericardium or peritoneum.
 18. The method of claim 17, wherein the collagen is type I collagen.
 19. A biocompatible collagen-coated serous membrane prepared by the method of claim
 7. 20. A biocompatible collagen-coated serous membrane prepared by the method of claim
 18. 